Methods of treatment with asparaginase

ABSTRACT

The invention relates to methods of treating diseases with L-asparaginase.

FIELD OF THE INVENTION

The present invention concerns a conjugate of a protein havingsubstantial L-asparagine aminohydrolase activity and polyethyleneglycol, particularly wherein the polyethylene glycol has a molecularweight less than or equal to about 5000 Da, particularly a conjugatewherein the protein is a L-asparaginase from Erwinia, and its use intherapy.

BACKGROUND OF THE INVENTION

Proteins with L-asparagine aminohydrolase activity, commonly known asL-asparaginases, have successfully been used for the treatment of AcuteLymphoblastic Leukemia (ALL) in children for many years. ALL is the mostcommon childhood malignancy (Avramis and Panosyan, (2005) 44:367-393).

L-asparaginase has also been used to treat Hodgkin's disease, acutemyelocytic Leukemia, acute myclomonocytic Leukemia, chronic lymphocyticLeukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia(2007) J. Biotechnol. 127, 657-669). The anti-tumor activity ofL-asparaginase is believed to be due to the inability or reduced abilityof certain malignant cells to synthesize L-asparagine (Kotzia (2007) J.Biotechnol. 127, 657-669). These malignant cells rely on anextracellular supply of L-asparagine. However, the L-asparaginase enzymecatalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia,thereby depleting circulating pools of L-asparagine and killing tumorcells which cannot perform protein synthesis without L-asparagine(Kotzia (2007) J. Biotechnol. 127, 657-669).

L-asparaginase from E. coli was the first enzyme drug used in ALLtherapy and has been marketed as Elspar® in the United States or asKidrolase® and L-asparaginase Medac® in Europe. L-asparaginases havealso been isolated from other microorganisms, e.g., an L-asparaginaseprotein from Erwinia chrysanthemi, named crisantaspase, that has beenmarketed as Erwinase® (Wriston (1985) Meth. Enzymol. 113, 608-618;Goward (1992) Bioseparation 2, 335-341). L-asparaginases from otherspecies of Erwinia have also been identified, including, for example,Erwinia chrysanthemi 3937 (Genbank Accession No. AAS67028), Erwiniachrysanthemi NCPPB 1125 (Genbank Accession No. CAA31239), Erwiniacarotovora (Genbank Accession No. AAP92666), and Erwinia carotovorasubsp. astroseptica (Genbank Accession No. AAS67027). These Erwiniachrysanthemi L-asparaginases have about 91-98% amino acid sequenceidentity with each other, while the Erwinia carotovora L-asparaginaseshave approximately 75-77% amino acid sequence identity with the Erwiniachrysanthemi L-asparaginases (Kotzia (2007) J. Biotechnol. 127 657-669).

The currently available L-asparaginase preparations do not providealternative or complementary therapies, particularly therapies to treatALL, that are characterized by high catalytic activity and significantlyimproved pharmacological and pharmacokinetic properties, as well asreduced immunogenicity.

In one aspect, the problem to be solved by the invention is to providean L-asparaginase preparation with: high in vitro bioactivity; a stablePEG-protein linkage; prolonged in vivo half-life; significantly reducedimmunogenicity, as evidenced, for example, by the reduction orelimination of an antibody response against the L-asparaginasepreparation following repeated administrations; and usefulness as asecond-line therapy for patients who have developed sensitivity tofirst-line therapies using, e.g., E. coli-derived L-asparaginases.

This problem has not been solved by known L-asparaginase conjugates,which either have significant cross-reactivity with modifiedL-asparaginase preparations (Wang (2003) Leukemia 17, 1583-1588incorporated herein by reference in its entirety), or which haveconsiderably reduced in vitro activity (Kuchumova (2007) Biochemistry(Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232incorporated herein by reference in its entirety). This problem issolved according to the present invention by providing a conjugate ofErwinia L-asparaginase with a hydrophilic polymer, more specifically, apolyethylene glycol with a molecular weight of 5000 Da or less, a methodfor preparing such a conjugate and the use of the conjugate.

SUMMARY OF THE INVENTION

The invention encompasses a method of treating a disease treatable byL-asparagine depletion in a patient comprising administering aneffective amount conjugate of a protein having substantial L-asparagineaminohydrolase activity and polyethylene glycol (PEG), wherein thepolyethylene glycol has a molecular weight less than or equal to about5000 Da, wherein the protein is a L-asparaginase from Erwinia. In someembodiments, the L-asparaginase has at least about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid of SEQ ID NO: 1. In some embodiments, the conjugate comprisesan L-asparaginase from Erwinia having at 100% sequence identity to theamino acid of SEQ ID NO: 1. In some embodiments, the PEG has a molecularweight of about 5000 Da, 4000, Da, 3000 Da, 2500 Da, or 2000 Da. In someembodiments, the conjugate has an in vitro activity of at least 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%as compared to the L-asparaginase when not conjugated to PEG. In someembodiments, the conjugate has an L-asparagine depletion activity atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more potentthan the L-asparaginase when not conjugated to PEG. In some embodiments,the conjugate depletes plasma L-asparagine levels to an undetectablelevel for at least about 12, 24, 48, 96, 108, or 120 hours. In someembodiments, the conjugate has a longer in vivo circulating half-lifecompared to the L-asparaginase when not conjugated to PEG. In someembodiments, the conjugate has a longer t½ than pegaspargaseadministered at an equivalent protein dose. In some embodiments, theconjugate has a t½ of at least about 58 to about 65 hours at a dose ofabout 50 μg/kg on a protein content basis, and a t½ of at least about 34to about 40 hours at a dose of about 10 μg/kg on a protein contentbasis, following iv administration in mice. In some embodiments, theconjugate has a t½ of at least about 100 to about 200 hours at a doseranging from about 10,000 to about 15,000 IU/m² (about 20-30 mgprotein/m²). In some embodiments, the conjugate has a greater area underthe curve (AUC) compared to the L-asparaginase when not conjugated toPEG. In some embodiments, the conjugate has a mean AUC that is at leastabout 3 times greater than pegaspargase at an equivalent protein dose.In some embodiments, the PEG is covalently linked to one or more aminogroups of the L-asparaginase. In some embodiments, the PEG is covalentlylinked to the one or more amino groups by an amide bond. In someembodiments, the PEG is covalently linked to at least from about 40% toabout 100% of the accessible amino groups or at least from about 40% toabout 90% of total amino groups.

The method of the invention encompass use of conjugate having theformula:

Asp-[NH—CO—(CH₂)_(x)—CO—NH-PEG]_(n)

wherein Asp is the L-asparaginase, NH is one or more of the NH groups ofthe lysine residues and/or the N-terminus of the Asp, PEG is apolyethylene glycol moiety, n is a number that represents at least about40% to about 100% of the accessible amino groups in the Asp, and x is aninteger ranging from about 1 to about 8, more specifically, from about 2to about 5. In a specific embodiment, the PEG ismonomethoxy-polyethylene glycol (mPEG).

The method of the invention encompass use of a conjugate ofL-asparaginase which comprises one or more peptide(s), wherein each isindependently a peptide R^(N)-(P/A)-R^(C), wherein (P/A) is an aminoacid sequence consisting solely of proline and alanine amino acidresidues, wherein R^(N) is a protecting group attached to the N-terminalamino group of the amino acid sequence, and wherein R^(C) is an aminoacid residue bound via its amino group to the C-terminal carboxy groupof the amino acid sequence, wherein each peptide is conjugated to theL-asparaginase via an amide linkage formed from the carboxy group of theC-terminal amino acid residue R^(C) of the peptide and a free aminogroup of the L-asparaginase, and wherein at least one of the free aminogroups, which the peptides are conjugated to, is not an N-terminalα-amino group of the L-asparaginase.

The method of the invention encompass use of the conjugate for thetreatment of cancer. In some embodiments, the cancer is selected fromthe group consisting of lymphoma, large cell immunoblastic lymphoma,non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, NK lymphoma,Hodgkin's disease, acute myelocytic Leukemia, acute promyelocyticLeukemia, acute myelomonocytic Leukemia, acute monocytic Leukemia, acuteT-cell Leukemia, acute myeloid Leukemia (AML), biphenotypic B-cellmyelomonocytic Leukemia and chronic lymphocytic Leukemia.

In some embodiments, the disease is selected from the group consistingof renal cell carcinoma, renal cell adenocarcinoma, glioblastomaincluding glioblastoma multiforma and glioblastoma astrocytoma,medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoidcarcinoma, squamous cell carcinoma, lung carcinoma including large celllung carcinoma and small cell lung carcinoma, endometrial carcinoma,ovarian adenocarcinoma, ovarian tetratocarcinoma, cervicaladenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductalcarcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, coloncarcinoma, colon adenocarcinoma, colorectal adenocarcinoma, bladdertransitional cell carcinoma, bladder papilloma, prostate carcinoma,osteosarcoma, epitheloid carcinoma of the bone, prostate carcinoma, andthyroid cancer. In some embodiments, the conjugate is administered at anamount of about 5 U/kg body weight to about 50 U/kg body weight.

In some embodiments, the conjugate is administered at a dose rangingfrom about 100 to about 15,000 IU/m². In some embodiments, theadministration is intravenous or intramuscular and is once per week,twice per week, or three times per week. In some embodiments, conjugateis administered as monotherapy. In some embodiments, the conjugate isadministered as part of a combination therapy. In some embodiments, theconjugate is administered as part of a combination therapy withOncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, Venetoclax,dactolisib, bortezomib, carfilzomib, vincristine, prednisolone,everolimus, and/or CB-839. In some embodiments, the patient receivingtreatment has had a previous hypersensitivity to an E. coli asparaginaseor PEGylated form thereof or to an Erwinia asparaginase. In someembodiments, the patient receiving treatment has had a disease relapse,in particular a relapse that occurs after treatment with an E. coliasparaginase or PEGylated form thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 and FIG. 2 depict in vivo experimental data usingpegcrisantaspase with other compounds.

FIG. 3 depicts dose-response curves with exemplary single agents.

FIG. 4 depicts dose-response curves with exemplary mixtures withinactive agents

FIG. 5 depicts comparison data for the exemplary single agents andmixtures.

FIG. 6 depicts a dose-oriented plot indicating whether drug combinationsare synergistic.

FIG. 7 depicts CNS cell line data.

FIG. 8 and FIG. 9 depict IC₅₀ effect of pegcrisantaspase.

FIG. 10 depicts in vitro sensitivity of pegcrisantaspase in leukemia andlymphoma cell lines.

FIG. 11A-FIG. 11C depicts mPEG-r-crisantaspase conjugate(Pegcrisantaspase) tested against various cell lines.

FIG. 12 depicts the results of a T-test.

FIG. 13A-FIG. 13B depicts mPEG-r-crisantaspase conjugate(Pegcrisantaspase and Oncaspar®) tested with other agents used in thestandard of care for AML or DLBCL.

FIG. 14A-FIG. 14B depicts Pegcrisantaspase combined with referencecompounds in AML and DLBCL cell lines.

FIG. 15 depicts testing of Erwinase in Oncolines™ and testing ofOncaspar in Oncolines™.

FIG. 16 depicts testing of PEG-crisantaspace Oncolines™.

FIG. 17 depicts testing of PA-20 Corynebacterium in Oncolines™ andtesting of PA-20 Corynebacterium in Oncolines™.

FIG. 18 depicts testing of PA-20 Psuedomonas in Oncolines™.

FIG. 19 depicts testing of PA-40 Psuedomonas in Oncolines™ and PA-200Psuedomonas in Oncolines™.

DETAILED DESCRIPTION OF THE INVENTION

L-asparaginases of bacterial origin have a high immunogenic andantigenic potential and frequently provoke adverse reactions rangingfrom mild allergic reaction to anaphylactic shock in sensitized patients(Wang (2003) Leukemia 17, 1583-1588). E. coli L-asparaginase isparticularly immunogenic, with reports of the presence ofanti-asparaginase antibodies to E. coli L-asparaginase following i.v. ori.m. administration reaching as high as 78% in adults and 70% inchildren (Wang (2003) Leukemia 17, 1583-1588).

L-asparaginases from Escherichia coli and Erwinia chrysanthemi differ intheir pharmacokinetic properties and have distinct immunogenic profiles,respectively (Klug Albertsen (2001) Brit. J. Haematol. 115, 983-990).Furthermore, it has been shown that antibodies that developed after atreatment with L-asparaginase from E. coli do not cross react withL-Asparaginase from Erwinia (Wang (2003) Leukemia 17, 1583-1588). Thus,L-asparaginase from Erwinia crisantaspase has been used as a second linetreatment of ALL in patients that react to E. coli L-asparaginase (Duval(2002) Blood 15, 2734-2739; Avramis (2005) Clin. Pharmacokinet. 44,367-393).

In another attempt to reduce immunogenicity associated withadministration of microbial L-asparaginases, an E. coli L-asparaginasehas been developed that is modified with methoxy-polyethyleneglycol(mPEG). This method is commonly known as “PEGylation” and has been shownto alter the immunological properties of proteins (Abuchowski (1977) J.Biol. Chem. 252, 3578-3581). This so-called mPEG-L-asparaginase, orpegaspargase, marketed as Oncaspar® was first approved in the U.S. forsecond line treatment of ALL in 1994, and has been approved forfirst-line therapy of ALL in children and adults since 2006. Oncaspar®has a prolonged in vivo half-life and a reducedimmunogenicity/antigenicity.

Oncaspar® is E. coli L-asparaginase that has been modified at multiplelysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S.Pat. No. 4,179,337). SS-PEG is a PEG reagent of the first generationthat contains an instable ester linkage that is sensitive to hydrolysisby enzymes or at slightly alkaline pH values (U.S. Pat. No. 4,670,417).These properties decrease both in vitro and in vivo stability and canimpair drug safety.

Furthermore, it has been demonstrated that antibodies developed againstL-asparaginase from E. coli will cross react with Oncaspar® (Wang (2003)Leukemia 17, 1583-1588). Even though these antibodies were notneutralizing, this finding clearly demonstrated the high potential forcross-hypersensitivity or cross-inactivation in vivo. Indeed, in onereport 30-41% of children who received pegaspargase had an allergicreaction (Wang (2003) Leukemia 17, 1583-1588).

In addition to outward allergic reactions, the problem of “silenthypersensitivity” was recently reported, whereby patients developanti-asparaginase antibodies without showing any clinical evidence of ahypersensitivity reaction (Wang (2003) Leukemia 17, 1583-1588). Thisreaction can result in the formation of neutralizing antibodies to E.coli L-asparaginase and pegaspargase; however, these patients are notswitched to Erwinia L-asparaginase because there are not outward signsof hypersensitivity, and therefore they receive a shorter duration ofeffective treatment (Holcenberg (2004) Pediatr. Hematol. Oncol. 26,273-274).

Erwinia chrysanthemi L-asparaginase treatment is often used in the eventof hypersensitivity to E. coli-derived L-asparaginases. However, it hasbeen observed that as many as 30-50% of patients receiving ErwiniaL-asparaginase arc antibody-positive (Avramis (2005) Clin.Pharmacokinet. 44, 367-393). Moreover, because Erwinia chrysanthemiL-asparaginase has a significantly shorter elimination half-life thanthe E. coli L-asparaginases, it must be administered more frequently(Avramis (2005) Clin. Pharmacokinet. 44, 367-393). In a study byAvramis, Erwinia asparaginase was associated with inferiorpharmacokinetic profiles (Avramis (2007) J. Pediatr. Hematol. Oncol. 29,239-247). E. coli L-asparaginase and pegaspargase therefore have beenthe preferred first-line therapies for ALL over Erwinia L-asparaginase.

Numerous biopharmaceuticals have successfully been PEGylated andmarketed for many years. In order to couple PEG to a protein, the PEGhas to be activated at its OH terminus. The activation group is chosenbased on the available reactive group on the protein that will bePEGylated. In the case of proteins, the most important amino acids arelysine, cysteine, glutamic acid, aspartic acid, C-terminal carboxylicacid and the N-terminal amino group. In view of the wide range ofreactive groups in a protein nearly the entire peptide chemistry hasbeen applied to activate the PEG moiety. Examples for this activatedPEG-reagents are activated carbonates, e.g., p-nitrophenyl carbonate,succinimidyl carbonate; active esters, e.g., succinimidyl ester; and forsite specific coupling aldehydes and maleimides have been developed(Harris (2002) Adv. Drug Del. Rev. 54, 459-476). The availability ofvarious chemical methods for PEG modification shows that each newdevelopment of a PEGylated protein will be a case by case study. Inaddition to the chemistry the molecular weight of the PEG that isattached to the protein has a strong impact on the pharmaceuticalproperties of the PEGylated protein. In most cases it is expected that,the higher the molecular weight of the PEG, the better the improvementof the pharmaceutical properties (Sherman (2008) Adv. Drug Del. Rev. 60,59-68; Holtsberg (2002) Journal of Controlled Release 80, 259-271). Forexample, Holtsberg et al. found that, when PEG was conjugated toarginine deaminase, another amino acid degrading enzyme isolated from amicrobial source, pharmacokinetic and pharmacodynamic function of theenzyme increased as the size of the PEG attachment increased from amolecular weight of 5000 Da to 20,000 Da (Holtsberg (2002) Journal ofControlled Release 80, 259-271).

However, in many cases, PEGylated biopharmaceuticals show significantlyreduced activity compared to the unmodified biopharmaceutical (Fishburn(2008) J. Pharm. Sci., 1-17). In the case of L-asparaginase from Erwiniacarotovora, it has been observed that PEGylation reduced its in vitroactivity to approximately 57% (Kuchumova (2007) Biochemistry (Moscow)Supplement Series B: Biomedical Chemistry, 1, 230-232). TheL-asparaginase from Erwinia carotovora has only about 75% homology tothe Erwinia chrysanthemi L-asparaginase (crisantaspase). For Oncaspar®it is also known that its in vitro activity is approximately 50%compared to the unmodified E. coli L-asparaginase.

Described herein is a PEGylated L-asparaginase from Erwinia withimproved pharmacological properties as compared with the unmodifiedL-asparaginase protein, as well as compared to the pegaspargasepreparation from E. coli. The PEGylated L-asparaginase conjugatedescribed herein, e.g., Erwinia chrysanthemi L-asparaginase PEGylatedwith 5000 Da molecular weight PEG, serves as a therapeutic agentparticularly for use in patients who show hypersensitivity (e.g., anallergic reaction or silent hypersensitivity) to treatment withL-asparaginase or PEGylated L-asparaginase from E. coli. or unmodifiedL-asparaginase from Erwinia. The PEGylated L-asparaginase conjugatedescribed herein is also useful as a therapeutic agent for use inpatients who have had a disease relapse, e.g., a relapse of ALL, andhave been previously treated with another form of asparaginase, e.g.,with L-asparaginase or PEGylated L-asparaginase from E. coli.

As described in detail herein, the conjugate of the invention showsunexpectedly superior properties compared to known L-asparaginasepreparations such as pegaspargase. For example, unmodifiedL-asparaginase from Erwinia chrysanthemi (crisantaspase) has asignificantly lower half-life than unmodified L-asparaginase from E.coli (Avramis (2005) Clin. Pharmacokinet. 44, 367-393 incorporatedherein by reference in its entirety). The PEGylated conjugate of theinvention has a half-life that is greater than PEGylated L-asparaginasefrom E. coli at an equivalent protein dose.

Definitions

Unless otherwise expressly defined, the terms used herein will beunderstood according to their ordinary meaning in the art.

As used herein, the term “including” means “including, withoutlimitation,” and terms used in the singular shall include the plural,and vice versa, unless the context dictates otherwise.

As used herein, the term “disease treatable by depletion of asparagine”refers to a condition or disorder wherein the cells involved in orresponsible for the condition or disorder either lack or have a reducedability to synthesize L-asparagine. Depletion or deprivation ofL-asparagine can be partial or substantially complete (e.g., to levelsthat are undetectable using methods and apparatus that arc known in theart).

As used herein, the term “therapeutically effective amount” refers tothe amount of a protein (e.g., asparaginase or conjugate thereof),required to produce a desired therapeutic effect.

As used herein, the term “sequence identity” is used interchangeablywith “homology” and as such can have the same meaning where appropriate.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients to a human subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the human subject at the same time. Co-administration includessimultaneous administration in separate compositions, administration atdifferent times in separate compositions, or administration in acomposition in which two or more active pharmaceutical ingredients arepresent. Simultaneous administration in separate compositions andadministration in a composition in which both agents are present is alsoencompassed in the methods of the invention.

L-Asparaginase Protein

The protein according to the invention is an enzyme with L-asparagineaminohydrolase activity, namely an L-asparaginase.

Many L-asparaginase proteins have been identified in the art, isolatedby known methods from microorganisms. (See, e.g., Savitri (2003) IndianJ. Biotechnol 2, 184-194 incorporated herein by reference in itsentirety). The most widely used and commercially availableL-asparaginases are derived from E. coli or from Erwinia chrysanthemi,both of which share 50% or less structural homology. Within the Erwiniaspecies, typically 75-77% sequence identity was reported between Erwiniachrysanthemi and Erwinia carotovora-derived enzymes, and approximately90% sequence identity was found between different subspecies of Erwiniachrysanthemi (Kotzia G A, Labrou E, Journal of Biotechnology (2007)127:657-669, incorporated herein by reference in its entirety). Somerepresentative Erwinia L-asparaginases include, for example, thoseprovided in Table 1:

TABLE 1 Species Accession No. % Identity Erwinia chrysanthemi 3937AAS67028 91% Erwinia chrysanthemi NCPPB 1125 CAA31239 98% Erwiniacarotovora subsp. astroscptica AAS67027 75% Erwinia carotovora AAP9266677%

The sequences of the Erwinia L-asparaginases and the GenBank entries ofTable 1 are herein incorporated by reference. Preferred L-asparaginasesused in therapy are L-asparaginase isolated from E. coli and fromErwinia, specifically, Erwinia chrysanthemi.

The L-asparaginases may be native enzymes isolated from themicroorganisms. They can also be produced by recombinant enzymetechnologies in producing microorganisms such as E. coli. As examples,the protein used in the conjugate of the invention can be a protein formE. coli produced in a recombinant E. coli producing strain, of a proteinfrom an Erwinia species, particularly Erwinia chrysanthemi, produced ina recombinant E. coli producing strain.

Enzymes can be identified by their specific activities. This definitionthus includes all polypeptides that have the defined specific activityalso present in other organisms, more particularly in othermicroorganisms. Often enzymes with similar activities can be identifiedby their grouping to certain families defined as PFAM or COG. PFAM(protein family database of alignments and hidden Markov models;pfam.sanfferac.ukl) represents a large collection of protein sequencealignments. Each PFAM makes it possible to visualize multiplealignments, see protein domains, evaluate distribution among organisms,gain access to other databases, and visualize known protein structures.COGs (Clusters of Orthologous Groups of proteins;vv-ww.nebi.nlm.nih.gov/COG/) are obtained by comparing protein sequencesfrom 43 fully sequenced genomes representing 30 major phylogeneticlines. Each COG is defined from at least three lines, which permits theidentification of former conserved domains.

The means of identifying homologous sequences and their percentagehomology or sequence identity are well known to those skilled in theart, and include in particular the BLAST programs, which can be usedfrom the website blast.ncbi.olo.nih.gov/Blast.cgi with the defaultparameters indicated on that website. The sequences obtained can then beexploited (e.g., aligned) using, for example, the programs CLUSTALW(www.ebi.ac.uk/Tools/clustalw2/index.html) or MULTALIN(bioinfo.genotoul.fr/multalin/multalin.html) with the default parametersindicated on those websites. Using the references given on GenBank forknown genes, those skilled in the art are able to determine theequivalent genes in other organisms, bacterial strains, yeasts, fungi,mammals, plants, etc. This routine work is advantageously done usingconsensus sequences that can be determined by carrying out sequencealignments with genes derived from other microorganisms, and designingdegenerate probes to clone the corresponding gene in another organism.These routine methods of molecular biology are well known to thoseskilled in the art, and are described, for example, in Sambrook (2012)Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor LabPress).

Indeed, a person skilled in the art will understand how to select anddesign homologous proteins retaining substantially their L-asparaginaseactivity. Typically, a Nessler assay is used for the determination ofL-asparaginase activity according to a method described by Mashburn andWriston (Mashburn (1963) Biochem. Biophys. Res. Comm. 12, 50incorporated herein by reference in its entirety).

In a particular embodiment of the conjugate of the invention, theL-asparaginase protein has at least about 80% homology or sequenceidentity with the protein comprising the sequence of SEQ ID NO: 1, morespecifically at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 20 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with theprotein comprising the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is asfollows:

ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGV DTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLH LTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMWLNDRIGSARYITKTNASTLDTF KANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVY AGIVIGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTS DPKVIQEYFHTY

The term “comprising the sequence of SEQ ID NO: 1” means that theamino-acid sequence of the protein may not be strictly limited to SEQ IDNO: 1 but may contain additional amino-acids.

In a particular embodiment, the protein is the L-asparaginase of Erwiniachrysanthemi having the sequence of SEQ ID NO: 1. In another embodiment,the L-asparaginase is from Erwinia chrysanthemi NCPPB 1066 (GenbankAccession No. CAA32884 incorporated herein by reference in itsentirety), either with or without signal peptides and/or leadersequences.

Fragments of the protein of SEQ ID NO: 1 are also comprised within thedefinition of the protein used in the conjugate of the invention. Theterm “a fragment of SEQ ID NO: 1” means that the sequence of thepolypeptide may include less amino-acid than SEQ ID NO: 1 but stillenough amino-acids to confer L-aminohydrolase activity.

It is well known in the art that a polypeptide can be modified bysubstitution, insertion, deletion and/or addition of one or moreamino-acids while retaining its enzymatic activity. For example,substitution of one amino-acid at a given position by a chemicallyequivalent amino-acid that does not affect the functional properties ofa protein is common. Substitutions may be defined as exchanges withinone of the following groups:

Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr,Pro, Gly;

Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln;

Polar, positively charged residues: His, Arg, Lys;

Large aliphatic, non-polar residues: Met, Leu, Ile, Val, Cys;

Large aromatic residues: Phe, Tyr, Trp.

Thus, changes that result in the substitution of one negatively chargedresidue for another (such as glutamic acid for aspartic acid) or onepositively charged residue for another (such as lysine for arginine) canbe expected to produce a functionally equivalent product.

The positions where the amino-acids are modified and the number ofamino-acids subject to modification in the amino-acid sequence are notparticularly limited. The skilled artisan is able to recognize themodifications that can be introduced without affecting the activity ofthe protein. For example, modifications in the N- or C-terminal portionof a protein may be expected not to alter the activity of a proteinunder certain circumstances. With respect to asparaginases, inparticular, much characterization has been done, particularly withrespect to the sequences, structures, and the residues forming theactive catalytic site. This provides guidance with respect to residuesthat can be modified without affecting the activity of the enzyme. Allknown L-asparaginases from bacterial sources have common structuralfeatures. All are homotetramers with four active sites between the N-and C-terminal domains of two adjacent monomers (Aghaipour (2001)Biochemistry 40, 5655-5664 incorporated herein by reference in itsentirety). All have a high degree of similarity in their tertiary andquaternary structures (Papageorgiou (2008) FEBS J. 275, 4306-4316incorporated herein by reference in its entirety). The sequences of thecatalytic sites of L-asparaginases are highly conserved between Erwiniachrysanthemi, Erwinia carotovora, and E. coli L-asparaginase II(Papageorgiou (2008) FEBS J. 275, 4306-4316). The active site flexibleloop contains amino acid residues 14-33, and structural analysis showthat Thr¹⁵, Thr⁹⁵, Ser⁶², Glu⁶³, Asp⁹⁶, and Ala¹²⁰ contact the ligand(Papageorgiou (2008) FEBS J. 275, 4306-4316). Aghaipour et al. haveconducted a detailed analysis of the four active sites of Erwiniachrysanthemi L-asparaginase by examining high resolution crystalstructures of the enzyme complexed with its substrates (Aghaipour (2001)Biochemistry 40, 5655-5664). Kotzia et. al provide sequences forL-asparaginases from several species and subspecies of Erwinia and, eventhough the proteins have only about 75-77% identity between Erwiniachrysanthemi and Erwinia carotovora, they each still have L-asparaginaseactivity (Kotzia (2007) J. Biotechnol. 127, 657-669 incorporated hereinby reference in its entirety). Moola et. al performed epitope mappingstudies of Erwinia chrysanthemi 3937 L-asparaginase and were able toretain enzyme activity even after mutating various antigenic sequencesin an attempt to reduce immunogenicity of the asparaginase (Moola (1994)Biochem. J. 302, 921-927 incorporated herein by reference in itsentirety). Each of the above-cited articles is herein incorporated byreference in its entirety. In view of the extensive characterizationthat has been performed on L-asparaginases, one of skill in the artcould determine how to make fragments and/or sequence substitutionswhile still retaining enzyme activity.

Polymers for Use in the Conjugate

Polymers are selected from the group of non-toxic water soluble polymerssuch as polysaccharides, e.g. hydroxyethyl starch, poly amino acids,e.g. poly lysine, polyester, e.g., polylactic acid, and poly alkyleneoxides, e.g., polyethylene glycol (PEG).

Polyethylene glycol (PEG) or mono-methoxy-polyethyleneglycol (mPEG) iswell known in the art and comprises linear and branched polymers.Examples of some polymers, particularly PEG, are provided in thefollowing, each of which is herein incorporated by reference in itsentirety: U.S. Pat. Nos. 5,672,662; 4,179,337; 5,252,714; U.S. PatentApplication Publication No. 2003/0114647; U.S. Pat. Nos. 6,113,906;7,419,600; 9,920,311 and PCT Publication No. WO2004/083258.

The quality of such polymers is characterized by the polydispersityindex (PDI). The PDI reflects the distribution of molecular weights in agiven polymer sample and is calculated from the weight average molecularweight divided by the number average molecular weight. It indicates thedistribution of individual molecular weights in a batch of polymers. ThePDI has a value always greater than 1, but as the polymer chainsapproach the ideal Gauss distribution (=monodispersity), the PDIapproaches 1.

The polyethylene glycol has advantageously a molecular weight comprisedwithin the range of about 500 Da to about 9,000 Da. More specifically,the polyethylene glycol (e.g, mPEG) has a molecular weight selected fromthe group consisting of polyethylene glycols of 2000 Da, 2500 Da, 3000Da, 3500 Da, 4000 Da, 4500 Da, and 5000 Da. In a particular embodiment,the polyethylene glycol (e.g., mPEG) has a molecular weight of 5000 Da.

Method for Preparing the Conjugate

For subsequent coupling of the polymer to proteins with L-asparagineaminohydrolase activity, the polymer moiety contains an activatedfunctionality that preferably reacts with amino groups in the protein.In one aspect, the invention is directed to a method of making aconjugate, the method comprising combining an amount of polyethyleneglycol (PEG) with an amount of L-asparaginase in a buffered solution fora time period sufficient to covalently link the PEG to theL-asparaginase. In a particular embodiment, the L-asparaginase is fromErwinia species, more specifically Erwinia chrysanthemi, and morespecifically, the L-asparaginase comprising the sequence of SEQ IDNO: 1. In one embodiment, the PEG is monomethoxy-polyethylene glycol(mPEG).

In one embodiment, the reaction between the polyethylene glycol andL-asparaginase is performed in a buffered solution. In some particularembodiments, the pH value of the buffer solution ranges between about7.0 and about 9.0. The most preferred pH value ranges between about 7.5and about 8.5, e.g., a pH value of about 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,8.1, 8.2, 8.3, 8.4, or 15 8.5. In a particular embodiment, theL-asparaginase is from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1.

Furthermore, PEGylation of L-asparaginase is performed at proteinconcentrations between about 0.5 and about 25 mg/mL, more specificallybetween about 2 and about 20 mg/mL and most specifically between about 3and about 15 mg/mL. For example, the protein concentration is about 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20mg/mL. In a particular embodiment, the PEGylation of L-asparaginase atthese protein concentrations is of Erwinia species, more specificallyErwinia chrysanthemi, and more specifically, the L-asparaginasecomprising the sequence of SEQ ID NO: 1.

At elevated protein concentrations of more than 2 mg/mL the PEGylationreaction proceeds rapidly, within less than 2 hours. Furthermore, amolar excess of polymer over amino groups in L-asparaginase of less thanabout 20:1 is applied. For example, the molar excess is less than about20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1,8:1, 7.5:1, 7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1,2:1, 1.5:1, or 1:1. In a specific embodiment the molar excess is lessthan about 10:1 and in a more specific embodiment, the molar excess isless than about 8:1. In a particular embodiment, the L-asparaginase isfrom Erwinia species, more specifically Erwinia chrysanthemi, and morespecifically, the L-asparaginase comprising the sequence of SEQ ID NO:1.

The number of PEG moieties which can be coupled to the protein will besubject to the number of free amino groups and, even more so, to whichamino groups are accessible for a PEGylation reaction. In a particularembodiment, the degree of PEGylation (i.e., the number of PEG moietiescoupled to amino groups on the L-asparaginase) is within a range fromabout 10% to about 100% of free and/or accessible amino groups (e.g.,about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).100% PEGylation of accessible amino groups (e.g., lysine residues and/orthe N-terminus of the protein) is also referred to herein as “maximallyPEGylated.” One method to determine the modified amino groups inmPEG-r-crisantaspase conjugates (degree of PEGylation) is a methoddescribed by Habeeb (A. F. S. A. Habeeb, “Determination of free aminogroups in proteins by trinitrobenzensulfonic acid”, Anal. Biochem. 14(1966), p. 328, incorporated herein by reference in its entirety). Inone embodiment, the PEG moieties are coupled to one or more amino groups(wherein amino groups include lysine residues and/or the N-terminus) ofthe L-asparaginase. In a particular embodiment, the degree of PEGylationis within a range of from about 10% to about 100% of total or accessibleamino groups (e.g., lysine residues and/or the N-terminus), e.g., about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100%. In a specific embodiment, about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total aminogroups (e.g., lysine residues and/or the N-terminus) are coupled to aPEG moiety. In another specific embodiment, about 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 70%, 71%, 72%,7%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%of the accessible amino groups (e.g., lysine residues and/or theN-terminus) are coupled to a PEG moiety. In a specific embodiment,40-55% or 100% of the accessible amino groups (e.g., lysine residuesand/or the N-terminus) are coupled to a PEG moiety. In some embodiments,the PEG moieties are coupled to the L-asparaginase by a covalentlinkage. In a particular embodiment, the L-asparaginase is from Erwiniaspecies, more specifically Erwinia chrysanthemi, and more specifically,the L-asparaginase comprising the sequence of SEQ ID NO: 1.

In one embodiment, the conjugate of the invention can be represented bythe formula

Asp-[NH—CO—(CH₂)_(x)—CO—NH-PEG]_(n)

wherein Asp is a L-asparaginase protein, NH is the NH group of a lysineresidue and/or the N-terminus of the protein chain, PEG is apolyethylene glycol moiety and n is a number of at least 40% to about100% of the accessible amino groups (e.g., lysine residues and/or theN-terminus) in the protein, all being defined above and below in theexamples, x is an integer ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6,7, 8), preferably 2 to 5 (e.g., 2, 3, 4, 5). In a particular embodiment,the L-asparaginase is from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1.

Other methods of PEGylation that can be used to form the conjugates ofthe invention are provided, for example, in U.S. Pat. Nos. 4,179,337,5,766,897, U.S. Patent Application Publication No. 2002/0065397A1, andU.S. Patent Application Publication No. 2009/0054590A1 each of which isherein incorporated by reference in its entirety.

Specific embodiments include proteins having substantial L-Asparagineaminohydrolase activity and polyethylene glycol, selected from the groupof conjugates wherein:

(A) the protein has at least 90% homology of structure with theL-asparaginase from Erwinia chrysanthemi as disclosed in SEQ ID NO: 1,the polyethylene glycol has a molecular weight of about 5000 Da, theprotein and polyethylene glycol moieties are covalently linked to theprotein by amide bonds, and about 100% of the accessible amino groups(e.g., lysine residues and/or the N-terminus) or about 80-90%, inparticular, about 84%, of total amino groups (e.g., lysine residuesand/or the N-terminus) are linked to a polyethylene glycol moiety.

(B) the protein has at least 90% homology with the L-asparaginase fromErwinia chrysanthemi as disclosed in SEQ ID NO: 1, the polyethyleneglycol has a molecular weight of about 5000 Da, the protein andpolyethylene glycol moieties are covalently linked to the protein byamide bonds, and about 40% to about 45%, and in particular about 43% ofthe accessible amino groups (e.g., lysine residues and/or theN-terminus), or about 36% of the total amino groups (e.g., lysineresidues and/or the N-terminus) arc linked to a polyethylene glycolmoiety.

(C) the protein has at least 90% homology with the L-asparaginase fromErwinia chrysanthemi as disclosed in SEQ ID NO: 1, the polyethyleneglycol has a molecular weight of about 2000 Da, the protein andpolyethylene glycol moieties are covalently linked to the protein byamide bonds, and about 100% of the accessible amino groups (e.g., one ormore lysine residues and/or the N-terminus) or about 80-90%, inparticular, about 84% of total amino groups (e.g., lysine residuesand/or the N-terminus) are linked to a polyethylene glycol moiety.

(D) the protein has at least 90% homology with the L-asparaginase fromErwinia chrysanthemi as disclosed in SEQ ID NO: 1, the polyethyleneglycol has a molecular weight of about 2000 Da, the protein andpolyethylene glycol moieties are covalently linked to the protein byamide bonds, and [0092] about 50% to about 60%, and in particular about55% of the accessible amino groups (e.g., lysine residues and/or theN-terminus) or about 47% of the total amino groups (e.g., lysineresidues and/or the N-terminus) are linked to a polyethylene glycolmoiety.

L-Asparaginase-PEG Conjugates

Conjugates of the invention have certain advantageous and unexpectedproperties compared to unmodified L-asparaginases, particularly comparedto unmodified Erwinia L-asparaginases, more particularly compared tounmodified L-asparaginase from Erwinia chrysanthemi, and moreparticularly compared to unmodified L-asparaginase having the sequenceof SEQ ID NO: 1.

In some embodiments, the methods of the invention encompass a conjugatewhich reduces plasma L-asparagine and glutamine levels for a time periodof at least about 12, 24, 48, 72, 96, or 120 hours when administered ata dose of 5 U/kg body weight (bw) or 10 μg/kg (protein content basis).In other embodiments, the conjugate of the invention reduces plasmaL-asparagine levels to undetectable levels for a time period of at leastabout 12, 24, 48, 72, 96, 120, or 144 hours when administered at a doseof 25 U/kg bw or 50 μg/kg (protein content basis). In other embodiments,the conjugate of the invention reduces plasma L-asparagine levels for atime period of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192,216, or 240 hours when administered at a dose of 50 U/kg bw or 100 μg/kg(protein content basis). In another embodiment, the conjugate of theinvention reduces plasma L-asparagine levels to undetectable levels fora time period of at least about 12, 24, 48, 72, 96, 120, 144, 168, 192,216, or 240 hours when administered at a dose ranging from about 100 toabout 15,000 IU/m² (about 1-30 mg protein/m²). In a particularembodiment, the conjugate comprises L-asparaginase from Erwinia species,more specifically Erwinia chrysanthemi, and more specifically, theL-asparaginase comprising the sequence of SEQ ID NO: 1. In a particularembodiment, the conjugate comprises PEG (e.g., mPEG) having a molecularweight of less than or equal to about 5000 Da. In a more particularembodiment, at least about 40% to about 100% of accessible amino groups(e.g., lysine residues and/or the N-terminus) are PEGylated.

In one embodiment, the conjugate comprises a ratio of mol PEG/molmonomer of about 4.5 to about 8.5, particularly about 6.5; a specificactivity of about 450 to about 550 U/mg, particularly about 501 U/mg;and a relative activity of about 75% to about 85%, particularly about81% compared to the corresponding unmodified L-asparaginase. In aspecific embodiment, the conjugate with these properties comprises anL-asparaginase from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1, with PEGylation of approximately 40-55%accessible amino groups (e.g., lysine residues and/or the N-terminus)with 5000 Da mPEG.

In one embodiment, the conjugate comprises a ratio of mol PEG/molmonomer of about 12.0 to about 18.0, particularly about 15.1; a specificactivity of about 450 to about 550 U/mg, particularly about 483 U/mg;and a relative activity of about 75 to about 85%, particularly about 78%compared to the corresponding unmodified L-asparaginase. In a specificembodiment, the conjugate with these properties comprises anL-asparaginase from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1, with PEGylation of approximately 100%accessible amino groups (e.g., lysine residues and/or the N-terminus)with 5000 Da mPEG.

In one embodiment, the conjugate comprises a ratio of mol PEG/molmonomer of about 5.0 to about 9.0, particularly about 7.0; a specificactivity of about 450 to about 550 U/mg, particularly about 501 U/mg;and a relative activity of about 80 to about 90%, particularly about 87%compared to the corresponding unmodified L-asparaginase. In a specificembodiment, the conjugate with these properties comprises anL-asparaginase from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1, with PEGylation of approximately 40-55%accessible amino groups (e.g., lysine residues and/or the N-terminus)with 10,000 Da mPEG.

In one embodiment, the conjugate comprises a ratio of mol PEG/molmonomer of about 11.0 to about 17.0, particularly about 14.1; a specificactivity of about 450 to about 550 U/mg, particularly about 541 U/mg;and a relative activity of about 80 to about 90%, particularly about 87%compared to the corresponding unmodified L-asparaginase. In a specificembodiment, the conjugate with these properties comprises anL-asparaginase from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1, with PEGylation of approximately 100%accessible amino groups (e.g., lysine residues and/or the N-terminus)with 10,000 Da mPEG.

In one embodiment, the conjugate comprises a ratio of mol PEG/molmonomer of about 6.5 to about 10.5, particularly about 8.5; a specificactivity of about 450 to about 550 U/mg, particularly about 524 U/mg;and a relative activity of about 80 to about 90%, particularly about 84%compared to the corresponding unmodified L-asparaginase. In a specificembodiment, the conjugate with these properties comprises anL-asparaginase from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1, with PEGylation of approximately 40-55%accessible amino groups (e.g., lysine residues and/or the N-terminus)with 2,000 Da mPEG.

In one embodiment, the conjugate comprises a ratio of mol PEG/molmonomer of about 12.5 to about 18.5, particularly about 15.5; a specificactivity of about 450 to about 550 U/mg, particularly about 515 U/mg;and a relative activity of about 80 to about 90%, particularly about 83%compared to the corresponding unmodified L-asparaginase. In a specificembodiment, the conjugate with these properties comprises anL-asparaginase from Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1, with PEGylation of approximately 100%accessible amino groups (e.g., lysine residues and/or the N-terminus)with 2,000 Da mPEG.

In other embodiments, the conjugate of the invention has an increasedpotency of at least about 10 times, 20 times, 30 times, 40 times, 50times, 60 times, 70 times, 80 times, 90 times, or 100 times after asingle injection compared to the corresponding unmodifiedL-asparaginase. In a specific embodiment, the conjugate with theseproperties comprises an L-asparaginase from Erwinia species, morespecifically Erwinia chrysanthemi, and more specifically, theL-asparaginase comprising the sequence of SEQ ID NO: 1. In a particularembodiment, the conjugate comprises PEG (e.g., mPEG) having a molecularweight of less than or equal to about 5000 Da. In a more particularembodiment, at least about 40% to about 100% of accessible amino groups(e.g., lysine residues and/or the N-terminus) are PEGylated.

In one embodiment, the conjugate of the invention has a single-dosepharmacokinetic profile determine as set forth in PCT Publication No.WO2011003886 according to the following, specifically wherein theconjugate comprises mPEG at molecular weight of less than or equal to2000 Da and an L-asparaginase from Erwinia species, more specificallyErwinia chrysanthemi, and more specifically, the L-asparaginasecomprising the sequence of SEQ ID NO: 1:

A_(max): about 150 U/L to about 250 U/L;

T_(Amax): about 4 h to about 8 h, specifically about 6 h;

d_(Amax): about 220 h to about 250 h, specifically, about 238.5 h (abovezero, from about 90 min to about 240 h);

AUC: about 12000 to about 30000; and

t½: about 50 h to about 90 h.

In one embodiment, the conjugate of the invention has a single-dosepharmacokinetic profile according to the following, specifically wherethe conjugate comprises mPEG at molecular weight of less than or equalto 5000 Da and an L-asparaginase from Erwinia species, more specificallyErwinia chrysanthemi, and more specifically, the L-asparaginasecomprising the sequence of SEQ ID NO: 1:

A_(max): about 18 U/L to about 250 U/L;

T_(Amax): about 1 h to about 50 h;

d_(Amax): about 90 h to about 250 h, specifically, about 238.5 h (abovezero, from about 90 min to about 240 h);

AUC: about 500 to about 35000; and

t½: about 30 h to about 120 h.

In one embodiment, the conjugate of the invention results in a similarlevel of L-asparagine depletion over a period of time (e.g., 24, 48, or72 hours) after a single dose compared to an equivalent quantity ofprotein of pegaspargase. In a specific embodiment, the conjugatecomprises an L-asparaginase from Erwinia species, more specificallyErwinia chrysanthemi, and more specifically, the L-asparaginasecomprising the sequence of SEQ ID NO: 1. In a particular embodiment, theconjugate comprises PEG (e.g., mPEG) having a molecular weight of lessthan or equal to about 5000 Da. In a more particular embodiment, atleast about 40% to about 100% of accessible amino groups (e.g., lysineresidues and/or the N-terminus) are PEGylated, more particularly about40-55% or 100%.

In one embodiment, the conjugate of the invention has a longer t½ thanpegaspargase administered at an equivalent protein dose. In a specificembodiment, the conjugate has a t½ of at least about 50, 52, 54, 56, 58,59, 60, 61, 62, 63, 64, or 65 hours at a dose of about 50 μg/kg (proteincontent basis). In another specific embodiment, the conjugate has a t½of at least about 30, 32, 34, 36, 37, 38, 39, or 40 hours at a dose ofabout 10 μg/kg (protein content basis). In another specific embodiment,the conjugate has a t½ of at least about 100 to about 200 hours at adose ranging from about 100 to about 15,000 IU/m² (about 1-30 mgprotein/m²).

In one embodiment, the conjugate of the invention has a mean AUC that isat least about 2, 3, 4 or 5 times greater than pegaspargase at anequivalent protein dose.

In one embodiment, the conjugate of the invention does not raise anysignificant antibody response for a particular period of time afteradministration of a single dose, e.g, greater than about 1 week, 2weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks,10 weeks, 11 weeks, 12 weeks, etc. In a particular embodiment theconjugate of the invention does not raise any significant antibodyresponse for at least 8 weeks. In one example, “does not raise anysignificant antibody response” means that the subject receiving theconjugate is identified within art-recognized parameters asantibody-negative. Antibody levels can be determined by methods known inthe art, for example ELISA or surface plasmon resonance (SPR-Biacore)assays (Zalewska-Szewczyk (2009) Clin. Exp. Med. 9,113-116; Avramis(2009) Anticancer Research 29, 299-302 each of which is incorporatedherein by reference in its entirety). Conjugates of the invention mayhave any combination of these properties.

PASylated L-asparaginase

In some embodiments, the methods of the invention encompass a conjugateof L-asparaginase which comprises one or more peptide(s), wherein eachis independently a peptide R^(N)-(P/A)-R^(C), wherein (P/A) is an aminoacid sequence consisting solely of proline and alanine amino acidresidues, wherein R^(N) is a protecting group attached to the N-terminalamino group of the amino acid sequence, and wherein R^(C) is an aminoacid residue bound via its amino group to the C-terminal carboxy groupof the amino acid sequence, wherein each peptide is conjugated to theL-asparaginase via an amide linkage formed from the carboxy group of theC-terminal amino acid residue R^(C) of the peptide and a free aminogroup of the L-asparaginase, and wherein at least one of the free aminogroups, which the peptides are conjugated to, is not an N-terminalα-amino group of the L-asparaginase. These molecules are also known asPASylated versions of L-asparaginase and are also referred to herein asconjugates.

The monomer of the modified L-asparaginase protein has from about 350,400, 450, 500, amino acids to about 550, 600, 650, 700, or 750 aminoacids after modification. In additional aspects, the modifiedL-asparaginase protein has from about 350 to about 750 amino acids, orabout 500 to about 750 amino acids.

Each peptide that is comprised in the modified L-asparaginase protein asdescribed herein is independently a peptide R^(N)-(P/A)-R^(C).Accordingly, for each of the peptides comprised in a modifiedL-asparaginase protein described herein, the N-terminal protecting groupR^(N), the amino acid sequence (P/A), and the C-terminal amino acidresidue R^(C) are each independently selected from their respectivemeanings. The two or more peptides comprised in the modifiedL-asparaginase protein may thus be the same, or they may be differentfrom one another. In one aspect, all of the peptides comprised in themodified L-asparaginase protein are the same.

The moiety (P/A) in the chemically conjugated modified L-asparaginaseprotein, which is comprised in the peptide R^(N)-(P/A)-R^(C), is anamino acid sequence that can consist of a total of between 10 to 100 ormore proline and alanine amino acid residues, a total of 15 to 60proline and alanine amino acid residues, a total of 15 to 45 proline andalanine amino acid residues, e.g. a total of 20 to about 40 proline andalanine amino acid residues, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, or 45 proline and alanine amino acid residues. In apreferred aspect, said amino acid sequence consists of about 20 prolineand alanine amino acid residues. In another preferred aspect, said aminoacid sequence consists of about 40 proline and alanine amino acidresidues. In the peptide R^(N)-(P/A)-R^(C), the ratio of the number ofproline residues comprised in the moiety (P/A) to the total number ofamino acid residues comprised in (P/A) is preferably ≥10% and ≤70%, morepreferably ≥20% and ≤50%, and even more preferably ≥25% and ≤40%.Accordingly, it is preferred that 10% to 70% of the total number ofamino acid residues in (P/A) are proline residues; more preferably, 20%to 50% of the total number of amino acid residues comprised in (P/A) areproline residues; and even more preferably, 25% to 40% (e.g., 25%, 30%,35% or 40%) of the total number of amino acid residues comprised in(P/A) are proline residues. Moreover, it is preferred that (P/A) doesnot contain any consecutive proline residues (i.e., that it does notcontain any partial sequence PP). In a preferred aspect, (P/A) is theamino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 2). In anotherpreferred aspect, (P/A) is the amino acid sequence

(SEQ ID NO: 3) AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA.

The group R^(N) in the peptide R^(N)-(P/A)-R^(C) may be a protectinggroup which is attached to the N-terminal amino group, particularly theN-terminal α-amino group, of the amino acid sequence (P/A). It ispreferred that R^(N) is pyroglutamoyl or acetyl.

The group R^(C) in the peptide R^(N)-(P/A)-R^(C) is an amino acidresidue which is bound via its amino group to the C-terminal carboxygroup of (P/A), and which comprises at least two carbon atoms betweenits amino group and its carboxy group. It will be understood that the atleast two carbon atoms between the amino group and the carboxy group ofR^(C) may provide a distance of at least two carbon atoms between theamino group and the carboxy group of R^(C) (which is the case if, e.g.,R^(C) is an ω-amino-C₃₋₁₅ alkanoic acid, such as ε-aminohexanoic acid).It is preferred that R^(C) is ε-aminohexanoic acid.

In one embodiment, the peptide is Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQID NO: 4) or Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQID NO: 5). The term “Pga” is an abbreviation of “pyroglutamoyl” or“pyroglutamic acid”. The term “Ahx” is an abbreviation of“ε-aminohexanoic acid”.

In the modified L-asparaginase proteins as described herein, eachpeptide R^(N)-(P/A)-R^(C), can be conjugated to the L-asparaginase viaan amide linkage formed from the carboxy group of the C-terminal aminoacid residue R^(C) of the peptide and a free amino group of theL-asparaginase. A free amino group of the L-asparaginase may be, e.g.,an N-terminal α-amino group or a side-chain amino group of theL-asparaginase (e.g., an ε-amino group of a lysine residue comprised inthe L-asparaginase). If the L-asparaginase is composed of multiplesubunits, e.g. if the L-asparaginase is a tetramer, there may bemultiple N-terminal α-amino groups (i.e., one on each subunit). In oneaspect, 9 to 13 peptides as defined herein (e.g. 9, 11, 12, or 13peptides) can be chemically conjugated to the L-asparaginase (e.g. toeach subunit/monomer of the L-asparaginase).

In accordance with the above, in one aspect at least one of the freeamino groups, which the peptides are chemically conjugated to, is not(i.e., is different from) an N-terminal α-amino group of theL-asparaginase. Accordingly, it is preferred that at least one of thefree amino groups, which the peptides are conjugated to, is a side-chainamino group of the L-asparaginase, and it is particularly preferred thatat least one of the free amino groups, which the peptides are conjugatedto, is an ε-amino group of a lysine residue of the L-asparaginase.

Moreover, it is preferred that the free amino groups, which the peptidesare conjugated to, are selected from the ε-amino group(s) of any lysineresidue(s) of the L-asparaginase, the N-terminal α-amino group(s) of theL-asparaginase or of any subunit(s) of the L-asparaginase, and anycombination thereof. It is particularly preferred that one of the freeamino groups, which the peptides are conjugated to, is an N-terminalα-amino group, while the other one(s) of the free amino groups, whichthe peptides are conjugated to, is/are each an ε-amino group of a lysineresidue of the L-asparaginase. Alternatively, it is preferred that eachof the free amino groups, which the peptides are conjugated to, is anε-amino group of a lysine residue of the L-asparaginase.

The modified L-asparaginase proteins as described herein are composed ofL-asparaginase and one or more peptides as defined herein. Acorresponding modified L-asparaginase protein may, e.g., consist of oneL-asparaginase and one, two, three, four, five, six, seven, eight, nine,ten, 15, 20, 25, 30, 35, 40, 45, 50, 55 (or more) peptides which areeach conjugated to the L-asparaginase. The L-asparaginase may be, e.g.,a monomeric protein or a protein composed of multiple subunits, e.g. atetramer. If the L-asparaginase is a monomeric protein, a correspondingmodified L-asparaginase protein may, e.g., consist of one monomericL-asparaginase and nine to thirteen (or more) (e.g., 8, 9, 10, 11, 12,or 13), peptides which are each conjugated to the monomericL-asparaginase. An exemplary amino acid sequence of a monomericL-asparaginase is shown in SEQ ID NO: 1. If the L-asparaginase is aprotein composed of multiple subunits, e.g. of four subunits (i.e. ifsaid L-asparaginase is a tetramer), a corresponding modifiedL-asparaginase protein may, e.g., consist of four L-asparaginasesubunits and nine to thirteen (or more) (e.g. 9, 10, 11, 12, or 13),peptides as defined herein which are each conjugated to each subunit ofthe L-asparaginase. An exemplary amino acid sequence of a subunit ofL-asparaginase is shown in SEQ ID NO. 1. Likewise, if the L-asparaginaseis a protein composed of multiple subunits, e.g. of four subunits (i.e.if said L-asparaginase is a tetramer), a corresponding modifiedL-asparaginase protein may, e.g., consist of four L-asparaginasesubunits and 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 (or more) peptides which areeach conjugated to the L-asparaginase tetramer. In one aspect theinvention relates to a modified L-asparaginase protein having anL-asparaginase and multiple chemically attached peptide sequences. In afurther aspect the length of the peptide sequences are from about 10 toabout 100, from about 15 to about 60 or from about 20 to about 40.

The peptide consisting solely of proline and alanine amino acid residuesmay be covalently linked to one or more amino acids of saidL-asparaginase, such as lysine residues and/or N-terminal residue,and/or the peptide consisting solely of proline and alanine amino acidresidues may be covalently linked to at least from about 40, 50, 60, 70,80 or 90% to about 60, 70, 80, 90 or 100% of the accessible amino groupsincluding amino groups of lysine residues and/or N-terminal residue onthe surface of the L-asparaginase. For example, there may be about 11 to12 lysine residues accessible per L-asparaginase, and about 8 to 12lysines would be conjugated to the peptide consisting solely of prolineand alanine amino acid residues. In further aspects, the peptideconsisting solely of proline and alanine amino acid residues iscovalently linked to from about 20, 30, 40, 50, or 60% to about 30, 40,50, 60, 70, 80 or 90% of total lysine residues of said L-asparaginase.In further embodiments, the peptide consisting solely of proline andalanine amino acid residues is covalently linked to the L-asparaginasevia a linker. Exemplary linkers include linkers disclosed in U.S. PatentApplication No. 2015/0037359, which is herein incorporated by referencein its entirety.

In one aspect, the conjugate is a fusion protein comprisingL-asparaginase and a polypeptide consisting solely of proline andalanine amino acid residues of a length of about 200 to about 400proline and alanine amino acid residues. In other words the polypeptidemay consist of about 200 to about 400 proline and alanine amino acidresidues. In one aspect, the polypeptide consists of a total of about200 (e.g. 201) proline and alanine amino acid residues (i.e. has alength of about 200 (e.g. 201) proline and alanine amino acid residues)or the polypeptide consists of a total of about 400 (e.g. 401) prolineand alanine amino acid residues (i.e. has a length of about 400 (e.g.401) proline and alanine amino acid residues). In some preferredembodiments, the polypeptide comprises or consists of an amino acidsequence as shown in SEQ ID NO: 6 or 7. In some aspects, the fusionprotein each monomer has from about 350, 400, 450, 500, amino acids toabout 550, 600, 650, 700, 750 or 1,000 amino acids including the monomerand the P/A amino acid sequence. In additional aspects, the modifiedprotein has from about 350 to about 800 amino acids or about 500 toabout 750 amino acids. For example, the polypeptide includes thepeptides prepared in U.S. Pat. No. 9,221,882. In some aspects, theL-asparaginase is from an Erwinia species, more specifically Erwiniachrysanthemi, and more specifically, the L-asparaginase comprising thesequence of SEQ ID NO: 1 as described herein.

In additional aspects, the L-asparaginase disclosed herein can beproduced using a (recombinant) vector comprising the nucleotide sequenceencoding the modified L-asaparaginase protein comprising theL-asparaginase and a polypeptide, wherein the polypeptide consistssolely of proline and alanine amino acid residues, preferably whereinthe modified protein is a fusion protein, as described herein, whereinthe vector can express the modified protein (e.g. fusion protein). Infurther aspects, the invention also relates to a host comprising the(recombinant) vector described herein. The host may be yeasts, such asSaccharomyces cerevisiae and Pichia Pistoris, bacteria, actinomycetes,fungi, algae, and other microorganisms, including Escherichia coli,Bacillus sp., Pseudomonas fluorescens, Corynebacterium glutamicum andbacterial hosts of the following genuses, Serratia, Proteus,Acinetobacter and Alcaligenes. Other hosts are known to those of skillin the art, including Nocardiopsis alba, which expresses a variant ofAsparaginase lacking on glutaminase-activity, and those disclosed inSavitri et al. (2003) Indian Journal of Biotechnology, 2, 184-194, whichis incorporated by reference herein in its entirety.

Methods of Treatment and Use

The conjugates s of the invention can be used in the treatment of adisease treatable by depletion of asparagine and/or glutamine. Forexample, the conjugate is useful in the treatment or the manufacture ofa medicament for use in the treatment of acute lymphoblastic Leukemia(ALL) in both adults and children, as well as other conditions whereasparagine and/or glutamine depletion is expected to have a usefuleffect. Such conditions include, but are not limited to the following:malignancies, or cancers, including but not limited to hematologicmalignancies, lymphoma, large cell immunoblastic lymphoma, non-Hodgkin'slymphoma, diffuse large B-cell lymphoma, NK lymphoma, Hodgkin's disease,acute myelocytic Leukemia, acute promyelocytic Leukemia, acutemyelomonocytic Leukemia, acute monocytic Leukemia, acute T-cellLeukemia, acute myeloid Leukemia (AML), biphenotypic B-cellmyelomonocytic Leukemia, chronic lymphocytic Leukemia, lymphosarcoma,reticulosarcoma, and melanosarcoma. In some embodiments, the disease maybe acute myeloid leukemia or diffuse large B-cell lymphoma. Malignanciesor cancers, include but not limited to, renal cell carcinoma, renal celladenocarcinoma, glioblastoma including glioblastoma multiforma andglioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignantmelanoma, epidermoid carcinoma, squamous cell carcinoma, lung carcinomaincluding large cell lung carcinoma and small cell lung carcinoma,endometrial carcinoma, ovarian adenocarcinoma, ovarian tetratocarcinoma,cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breastductal carcinoma, pancreatic adenocarcinoma, pancreatic ductalcarcinoma, colon carcinoma, colon adenocarcinoma, colorectaladenocarcinoma, bladder transitional cell carcinoma, bladder papilloma,prostate carcinoma, osteosarcoma, epitheloid carcinoma of the bone,prostate carcinoma, and thyroid cancer.

Representative non-malignant hematologic diseases which respond toasparagine and/or glutamine depletion include immune system-mediatedBlood diseases, e.g., infectious diseases such as those caused by HIVinfection (i.e., AIDS). Non-hematologic diseases associated withasparagine and/or glutamine dependence include autoimmune diseases, forexample rheumatoid arthritis, systemic lupus erythematosus (SLE),collagen vascular diseases, etc. Other autoimmune diseases includeosteo-arthritis, Issac's syndrome, psoriasis, insulin dependent diabetesmellitus, multiple sclerosis, sclerosing panencephalitis, rheumaticfever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn'sdisease), primary billiary cirrhosis, chronic active hepatitis,glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves'disease. Cells suspected of causing disease can be tested for asparagineand/or glutamine dependence in any suitable in vitro or in vivo assay,e.g., an in vitro assay wherein the growth medium lacks asparagineand/or glutamine. Thus, in one aspect, the invention is directed to amethod of treating a disease treatable in a patient, the methodcomprising administering to the patient an effective amount of aconjugate of the invention. In another aspect, the conjugate of theinvention is co-administered with another active pharmaceuticalingredient. In some embodiments, the conjugate of the invention isco-administered with Oncaspar®, daunorubicin, cytarabine, Vyxeos®,ABT-737, Venetoclax, dactolisib, bortezomib, carfilzomib, vincristine,prednisolone, everolimus, and/or CB-839. In a specific embodiment, thedisease is ALL. In a particular embodiment, the conjugate used in thetreatment of a disease treatable by asparagine and/or glutaminedepletion comprises an L-asparaginase from Erwinia species, morespecifically Erwinia chrysanthemi, and more specifically, theL-asparaginase comprising the sequence of SEQ ID NO: 1 as describedherein.

In one embodiment, treatment with a conjugate of the invention will beadministered as a first line therapy. In another embodiment, treatmentwith a conjugate of the invention will be administered as a second linetherapy in patients, particularly patients with ALL, where objectivesigns of allergy or hypersensitivity, including “silenthypersensitivity,” have developed to other asparaginase preparations, inparticular, the native Escherichia-coli-derived L-asparaginase or itsPEGylated variant (pegaspargase). Non-limiting examples of objectivesigns of allergy or hypersensitivity include testing “antibody positive”for an asparaginase enzyme. In a specific embodiment, the conjugate ofthe invention is used in second line therapy after treatment withpegaspargase. In a more specific embodiment, the conjugate used insecond line therapy comprises an L-asparaginase from Erwinia species,more specifically Erwinia chrysanthemi, and more specifically, theL-asparaginase comprising the sequence of SEQ ID NO: 1. In a morespecific embodiment, the conjugate further comprises PEG (e.g., mPEG)having a molecular weight of less than or equal to about 5000 Da, morespecifically about 5000 Da. In an even more specific embodiment, atleast about 40% to about 100% of accessible amino groups (e.g., lysineresidues and/or the N-terminus) are PEGylated, more particularly about40-55% or 100%.

In another aspect, the invention is directed to a method for treatingacute lymphoblastic leukemia comprising administering to a patient inneed of the treatment a therapeutically effective amount of a conjugateof the invention. In another aspect, the invention is directed to amethod for treating acute myeloid leukemia comprising co-administeringto a patient in need of the treatment a therapeutically effective amountof a conjugate of the invention in combination with daunorubicin,cytarabine, Vyxeos®, ABT-737, venetoclax, dactolisib, bortexomib, and/orcarfilzomib. In another aspect, the invention is directed to a methodfor treating acute myeloid leukemia comprising co-administering to apatient in need of the treatment a therapeutically effective amount of aconjugate of the invention in combination with venetoclax. In anotheraspect, the invention is directed to a method for treating diffuse largeB-cell lymphoma comprising co-administering to a patient in need of thetreatment a therapeutically effective amount of a conjugate of theinvention in combination with ABT-737, venetoclax, carfilzomib,vincristine, and/or prednisolone. In another aspect, the invention isdirected to a method for treating diffuse large B-cell lymphomacomprising co-administering to a patient in need of the treatment atherapeutically effective amount of a conjugate of the invention incombination with vincristine.

In another aspect, the conjugate described herein will be administeredat a dose ranging from about 1500 IU/m² to about 15,000 IU/m², typicallyabout 10,000 to about 15,000 IU/m² (about 20-30 mg protein/m²), at aschedule ranging from about twice a week to about once a month,typically once per week or once every other week, as a single agent(e.g., monotherapy) or as part of a combination of chemotherapy drugs,including, but not limited to glucocorticoids, corticostcroids,anticancer compounds or other agents, including, but not limited tomethotrexate, dexamethasone, prednisone, prednisolone, vincristine,cyclophosphamide, and anthracycline. As an example, patients with ALLwill be administered the conjugate of the invention as a component ofmulti-agent chemotherapy during chemotherapy phases including induction,consolidation or intensification, and maintenance. In a specificexample, the conjugate is not administered with an asparagine synthetaseinhibitor (e.g., such as set forth in U.S. Pat. No. 9,920,311 which isherein incorporated by reference in its entirety). In another specificexample, the conjugate is not administered with an asparagine synthetaseinhibitor, but is administered with other chemotherapy drugs. Theconjugate can be administered before, after, or simultaneously withother compounds as part of a multi-agent chemotherapy regimen.

In a specific embodiment, the method comprises administering a conjugateof the invention at an amount of about 1 U/kg to about 25 U/kg (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 U/kg) or an equivalent amount thereof 20(e.g., on a protein content basis). In a more specific embodiment, theconjugate is administered at an amount selected from the groupconsisting of about 5, about 10, and about 25 U/kg. In another specificembodiment, the conjugate is administered at a dose ranging from about1,000 IU/m² to about 20,000 IU/m² (e.g., 1,000 IU/m², 2,000 IU/m², 3,000IU/m², 4,000 IU/m², 5,000 IU/m², 6,000 IU/m², 7,000 IU/m², 8,000 IU/m²,9,000 IU/m², 10,000 IU/m², 11,000 IU/m², 12,000 IU/m², 13,000 IU/m²,14,000 IU/m², 15,000 IU/m², 16,000 IU/m², 17,000 IU/m², 18,000 IU/m²,19,000 IU/m², or 20,000 IU/m²). In another specific embodiment, theconjugate is administered at a dose that depletes L-asparagine and/orglutamine to undetectable levels using methods and apparatus known inthe art for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6,7, 8, 9, or 10 days) for a single dose.

In another embodiment, the method comprises administering a conjugate ofthe invention that elicits a lower immunogenic response in a patientcompared to an unconjugated L-asparaginase. In another embodiment, themethod comprises administering a conjugate of the invention that has alonger in vivo circulating half-life after a single dose compared to theunconjugated L-asparaginase. In one embodiment, the method comprisesadministering a conjugate that has a longer t½ than pegaspargaseadministered at an equivalent protein dose. In a specific embodiment,the method comprises administering a conjugate that has a t½ of at leastabout 50, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, or 65 hours at a doseof about 50 μg/kg (protein content basis). In another specificembodiment, the method comprises administering a conjugate that has a t½of at least about 30, 32, 34, 36, 37, 37, 39, or 40 hours at a dose ofabout 10 μg/kg (protein content basis). In another specific embodiment,the method comprises administering a conjugate that has a t½ at leastabout 100 to about 200 hours at a dose ranging from about 10,000 toabout 15,000 IU/IU/m² (about 20-30 mg protein/IU/m²). In one embodiment,the method comprises administering a conjugate that has a mean AUC thatis at least about 2, 3, 4 or 5 times greater than pegaspargase at anequivalent protein dose.

The incidence of relapse in ALL patients following treatment withL-asparaginase remains high, with approximately 10-25% of pediatric ALLpatients having early relapse (e.g. some during maintenance phase at30-36 month post-induction) (Avramis (2005) Clin. Pharmacokinet. 44,367-393). If a patient treated with E. coli-derived L-asparaginase has arelapse, subsequent treatment with E. coli preparations could lead to a“vaccination” effect, whereby the E. coli preparation has increasedimmunogenicity during the subsequent administrations. In one embodiment,the conjugate of the invention may be used in a method of treatingpatients with relapsed ALL who were previously treated with otherasparaginase preparations, in particular those who were previouslytreated with E. coli-derived asparaginases.

In some embodiments, the uses and methods of treatment of the inventioncomprise administering an L-asparaginase conjugate having properties orcombinations of properties described herein above (e.g., in the sectionentitled L-asparaginase PEG conjugates or PASylated L-asparaginase) orherein below.

Compositions, Formulations, and Routes of Administration

The invention also includes a pharmaceutical composition comprising aconjugate of the invention. In a specific embodiment, the pharmaceuticalcomposition is contained in a vial as a lyophilized powder to bereconstituted with a solvent, such as currently available nativeL-asparaginases, whatever the bacterial source used for its production(Kidrolase®, Elspar®, Erwinase®). In another embodiment, thepharmaceutical composition may further comprises a “ready to use”solution, such as pegaspargase (Oncaspar®) enabling, further to anappropriate handling, an administration through, e.g., intramuscular,intravenous (infusion and/or bolus), intra-cerebro-ventricular (icy),subcutaneous routes. In additional embodiments, the pharmaceuticalcomposition may comprise the conjugate of the invention in combinationwith Oncaspar®, daunorubicin, cytarabine, ABT-737, Venetoclax,dactolisib, bortezomib, carfilzomib, vincristine, prednisolone,everolimus, and/or CB-839.

Conjugates of the invention, including compositions comprisingconjugates of the invention (e.g., a pharmaceutical composition) can beadministered to a patient using standard techniques. Techniques andformulations generally may be found in Remington's PharmaceuticalSciences (2013) 22nd ed., Mack Publishing herein incorporated byreference.

Suitable dosage forms, in part, depend upon the use or the route ofentry, for example, oral, transdermal, transmucosal, or by injection(parenteral). Such dosage forms should allow the therapeutic agent toreach a target cell or otherwise have the desired therapeutic effect.For example, pharmaceutical compositions injected into the Blood streampreferably are soluble.

Conjugates and/or pharmaceutical compositions according to the inventioncan be formulated as pharmaceutically acceptable salts and complexesthereof. Pharmaceutically acceptable salts are non-toxic salts presentin the amounts and concentrations at which they are administered. Thepreparation of such salts can facilitate pharmaceutical use by alteringthe physical characteristics of the compound without preventing it fromexerting its physiological effect. Useful alterations in physicalproperties include lowering the melting point to facilitate transmucosaladministration and increasing solubility to facilitate administeringhigher concentrations of the drug. The pharmaceutically acceptable saltof an asparaginase may be present as a complex, as those in the art willappreciate.

Pharmaceutically acceptable salts include acid addition salts such asthose containing sulfate, hydrochloride, fumarate, maleate, phosphate,sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts canbe obtained from acids, including hydrochloric acid, maleic acid,sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid,lactic acid, tartaric acid, malonic acid, methanesulfonic acid,ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,cyclohexylsulfamic acid, fumaric acid, and quinic acid.

Pharmaceutically acceptable salts also include basic addition salts suchas those containing benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium, ammonium, alkylamine, and zinc, whenacidic functional groups, such as carboxylic acid or phenol are present.For example, see Remington's Pharmaceutical Sciences, supra. Such saltscan be prepared using the appropriate corresponding bases.

Pharmaceutically acceptable carriers and/or excipients can also beincorporated into a pharmaceutical composition according to theinvention to facilitate administration of the particular asparaginase.Examples of carriers suitable for use in the practice of the inventioninclude calcium carbonate, calcium phosphate, various sugars such aslactose, glucose, or sucrose, or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols, and physiologicallycompatible solvents. Examples of physiologically compatible solventsinclude sterile solutions of water for injection (WFI), saline solutionand dextrose.

Pharmaceutical compositions according to the invention can beadministered by different routes, including intravenous,intraperitoneal, subcutaneous, intramuscular, oral, topical(transdermal), or transmucosal administration. For systemicadministration, oral administration is preferred. For oraladministration, for example, the compounds can be formulated intoconventional oral dosage forms such as capsules, tablets, and liquidpreparations such as syrups, elixirs, and concentrated drops.

Alternatively, injection (parenteral administration) may be used, e.g.,intramuscular, intravenous, intraperitoneal, and subcutaneous injection.For injection, pharmaceutical compositions are formulated in liquidsolutions, preferably in physiologically compatible buffers orsolutions, such as saline solution, Hank's solution, or Ringer'ssolution. In addition, the compounds may be formulated in solid form andredissolved or suspended immediately prior to use. For example,lyophilized forms of the conjugate can be produced. In a specificembodiment, the conjugate is administered intramuscularly. In anotherspecific embodiment, the conjugate is administered intravenously.

Systemic administration can also be accomplished by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are well known in the art, and include, forexample, for transmucosal administration, bile salts, and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration, for example, may be throughnasal sprays, inhalers (for pulmonary delivery), rectal suppositories,or vaginal suppositories. For topical administration, compounds can beformulated into ointments, salves, gels, or creams, as is well known inthe art.

The amounts of the conjugate to be delivered will depend on manyfactors, for example, the IC₅₀, EC₅₀, the biological half-life of thecompound, the age, size, weight, and physical condition of the patient,and the disease or disorder to be treated. The importance of these andother factors to be considered are well known to those of ordinary skillin the art. Generally, the amount of the conjugate to be administeredwill range from about 10 International Units per square meter of thesurface area of the patient's body (IU/m²) to 50,000 IU/m², with adosage range of about 1,000 IU/m² to about 15,000 IU/m² being preferred,and a range of about 6,000 IU/m² to about 15,000 IU/ m² being morepreferred, and a range of about 10,000 to about 15,000 IU/m² (about20-30 mg protein/m²) being particularly preferred to treat a malignanthematologic disease, e.g., Leukemia. Typically, these dosages arcadministered via intramuscular or intravenous injection at an intervalof about 3 times weekly to about once per month, typically once per weekor once every other week during the course of therapy. Of course, otherdosages and/or treatment regimens may be employed, as determined by theattending physician.

This invention is further illustrated by the following additionalexamples that should not be construed as limiting. Those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made to the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

EXAMPLES

The subject matter of U.S. Pat. No. 9,920,311 is herein incorporated byreference including the Examples disclosing methods of producing andtesting PEGylated Asparaginase. The mPEG-r-crisantaspase conjugate usedin the following examples was prepared as set forth in U.S. Pat. No.9,920,311.

Example 1

mPEG-r-crisantaspase conjugate (Pegcrisantaspase) was tested againstvarious cell lines as shown in two stages in FIG. 11A.

Cell preparation. All cell lines have been licensed from the AmericanType Culture Collection (ATCC) Manassas, Va. (US). Master and WorkingCell banks (MCB and WCB) were prepared by subculturing inATCC-recommended media and freezing according to ATCC recommendedprotocols (www.atcc.org).

Compound preparation. Test compounds were prepared as stock solutions inDMSO or aqueous buffers as appropriate and serially diluted to obtain adilution series.

Cell proliferation assay. Cell proliferation was assessed using acommercially available luminescence assay using ATP as the endpoint.

Controls. t=0 signal. On a parallel plate, 45 μl cells were dispensedand incubated in a humidified atmosphere of 5% CO2 at 37° C. After 24hours 5 μl DMSO-containing Hepes buffer and 25 μl ATPlite 1Step™solution were mixed, and luminescence measured after 10 minuteincubation (=luminescencet=0).

Reference compound. The IC₅₀ of the reference compound doxorubicin ismeasured on a separate plate. The IC₅₀ is trended. If the IC₅₀ is out ofspecification (0.32-3.16 times deviating from historic average) theassay is invalidated.

Cell growth control. The cellular doubling times of all cell lines arecalculated from the t=0 hours and t=end growth signals of the untreatedcells. If the doubling time is out of specification (0.5-2.0 timesdeviating from historic average) the assay is invalidated.

Maximum signals. For each cell line, the maximum luminescence wasrecorded after incubation until t=end without compound in the presenceof 0.4% DMSO (=luminescence_(untreated,t=end)).

Drug sensitivity. The ‘log IC₅₀ differences between the “modified and“wild type’ groups of cell lines were analyzed in three ways. First, forthe eighteen most frequent genetic changes, drug sensitivities ofindividual cell lines were visualized in waterfall plots. Secondly, alarger subset of the most commonly occurring and best known cancer genes(38 in total) was analyzed with type II Anova analysis in thestatistical program R. The results are displayed in a volcano plot.Thirdly, the complete set of 114 cancer genes was analyzed by atwo-sided homoscedastic t-test in R. The p-values from Anova and t-testwere subjected to a Benjamini-Hochberg multiple testing correction, andonly genetic associations with a false discovery rate less than 20% areconsidered significant. The type II Anova analysis on 38 cancer genes isa different test than the homoscedastic t-test on 114 cancer genes,meaning that the significance of the associations may differ. For moreinformation on Oncolines™ methods see www.ntrc.nl/services/oncolinestm.

IC₅₀ were calculated by non-linear regression using IDBS XLfit. Thepercentage growth after incubation until t=end (%-growth) was calculatedas follows: 100%×(luminescence_(t=end)/luminescence_(untreated,t=end)).This was fitted to the ¹⁰log compound concentration (conc) by a4-parameter logistics curve:%-growth=bottom+(top−bottom)/(1+10^((logIC50-conc)*hill))) where hill isthe Hill-coefficient, and bottom and top the asymptotic minimum andmaximum cell growth that the compound allows in that assay.

NCI60 parameters. The LD₅₀, the concentration at which 50% of cells die,is the concentration where luminescence_(t=end)=½×luminescence_(t=0h).The GI₅₀, the concentration of 50% growth inhibition, is theconcentration where cell growth is half maximum. This is concentrationassociated with the signal:((luminescence_(untreated,t=end)−luminescence_(t=0))/2)+luminescence_(t=0).

Curve fitting. Curves calculated automatically by the software wereadjusted manually according to the following protocol: The curve bottomwas fixed at 0% when the calculated curve had a bottom below zero. Thehill was fixed on −6 when the software calculated a lower value. Curveswere invalidated when the F-test value for fitting quality was >1.5 orwhen the compound was inactive (<20% maximal effect), in which casescurves were removed from the graphs. When a curve had a biphasiccharacter, it was fitted on the most potent IC₅₀. Incidentally, whentechnical failures were likely, concentration points were knocked out.This is always shown in the dose-response graphs. The maximal effect(Max effect) was calculated as 100% (signal of untreated cells) minusthe curve bottom when the dose-response curve was completely determinedfor more than 85%. A dose-response curve is considered 100% completewhen the data points at the highest concentrations reach the curvebottom. If the completeness was smaller than 85%, Max effect wascalculated as 100% minus the average of the lowest signal. In caseswhere the bottom of the curve was locked on 0%, the maximal effect wasalways calculated as 100% minus the growth inhibition at the highestconcentration.

Volcano plot. The volcano plot in FIG. 8 shows how genetictransformations in 38 important genes are statistically associated withshifts in compound sensitivity (as measured by ¹⁰logIC₅₀). The p-value(y-axis in the volcano plot) indicates the confidence level for geneticassociation of mutations in a particular gene with a IC₅₀ shift. Thefactor of the IC₅₀ shift is indicated on the x-axis. The areas of thecircles are proportional to the number of mutants in the cell panel(each mutation is present at least three times). To computesignificance, p-values are subjected to a Benjamin-Hochberg multipletesting correction, and only genetic associations with a <20% falsediscovery rate are colored grey. The relevant cutoff p-value (0.059) isindicated by a horizontal line. If there are no significantassociations, no grey circles and horizontal line are drawn.

Results of the T-test. For 98 validated cancer driver genes, of whichmutations also occur in patients, it was tested if presence of ‘wildtype’ and ‘mutant’ variants of the gene in cell lines, is associatedwith a significant IC₅₀s shift of the investigated compound. The column‘IC₅₀ shift’ indicates the ¹⁰logIC₅₀ difference. A negative IC₅₀ shiftindicates that the compound is more potent in cell lines that carry the‘mutant’ gene. The column ‘p-value’ indicates the result of a two-sidedt-test. To compute significance, p-values were subjected to aBenjamin-Hochberg multiple testing correction, and only geneticassociations with a <20% false discovery rate are highlighted (column‘adj. p-value’). If there are no significant associations, there are nogrey cells in the table depicted in FIG. 12 .

The special volcano plot of FIG. 9 relates compound sensitivity (asmeasured by ¹⁰logIC₅₀) to the presence of cancer hotspot mutations. Thisprovides increased focus on clinically relevant cancer driver mutationsin comparison to the previous analyses. The hotspot mutations werederived from statistical analyses of the recurrence patterns ofmutations and copy number alterations in patients through separatestudies. Axes and statistical analyses are identical to the volcano plotof FIG. 8 . The cutoff p-level for significance is 0.32.Example 3: Synergistic activity of Pegcrisantaspase and Oncaspar®.Effect₂₀ For determination of the effect of the compound on the activityof other anti-cancer agents in SynergyScreen™ experiments, a low, fixedconcentration is used, corresponding to the concentration at which cellgrowth is inhibited by 20%. This concentration is determined using thedose-response curves of the single compounds. The concentration is thevalue on the x-as, corresponding to 80% viability of untreated at they-axis.

Oncaspar in Oncolines Cell line ATCC ref. Disease Effect₂₀ (IU/mL) KG-1CCL-246 Acute myelogenous leukemia (AML) 0.0001 HL-60 CCL-240 Acutepromyelocytic leukemia 0.0003 THP-1 TIB-202 Acute monocytic leukemia0.31 DB CRL-2289 Large cell lymphoma, B lymphoblast 0.47 HT CRL-2260Diffuse mixed lymphoma, B lymphoblast 0.28 RL CRL-2261 Non-Hodgkin'slymphoma, B lymphoblast 0.37 MOLT-4 CRL-1582 Acute lymphoblasticleukemia (ALL) 0.00019 U-87-MG HTB-14 Glioblastoma, brain 0.4 HT-1080CCL-121 Fibrosarcoma 0.00025 MV-4-11 CRL-9591 biphenotypic Bmyelomonocytic leukemia 0.26

Pegcrisantaspase in Oncolines Cell line ATCC ref. Disease Effect₂₀(IU/mL) KG-1 CCL-246 Acute myelogenous leukemia (AML) 0.00018 HL-60CCL-240 Acute promyelocytic leukemia 0.00028 THP-1 TIB-202 Acutemonocytic leukemia 0.012 DB CRL-2289 Large cell lymphoma, B lymphoblast0.059 HT CRL-2260 Diffuse mixed lymphoma, B lymphoblast 0.033 RLCRL-2261 Non-Hodgkin's lymphoma, B lymphoblast 0.037 MOLT-4 CRL-1582Acute lymphoblastic leukemia (ALL) 0.00019 U-87-MG HTB-14 Glioblastoma,brain 0.057 HT-1080 CCL-121 Fibrosarcoma 0.013 MV-4-11 CRL-9591biphenotypic B myelomonocytic leukemia 0.0088mPEG-r-crisantaspase conjugate (Pegcrisantaspase; see FIG. 13A) orOncaspar® (see FIG. 13B) were tested with other agents that aretypically used in the standard of care (SOC) for AML or DLBCL. There wasan increased effect in AML when used with daunorubicin, cytarabine,ABT-737, Venetoclax, dactolisib, bortezomib, and carfilzomib.Additionally, there was an increased effect in DLBCL when used withvincristine, prednisolone, ABT-737, venetoclax, everolimus, dactolisib,bortezomib, carfilzomib, and CB-839. See the tables depicted in FIG.13A-FIG. 13B. Grey shading indicates synergistic activity. Light greyshading indicates one experiment and dark grey indicates twoexperiments.Example 3: mPEG-r-crisantaspase conjugates (Pegcrisantaspases) weretested in vivo with cytarabine and daunorubicin. Groups of 5 mice eachwere given mPEG-r-crisantaspase (PegC) as a single agent (5 & 50 IU/kg)and given in combination with SOC agent cytarabine (50 mg/kg once a dayfor 5 days followed by 2 days rest for 2 cycles) and daunorubicin (1mg/kg administered weekly for 2 weeks). These doses were well tolerated.See FIG. 1 . Group 1 is PBS control, Group 3 is PegC, Group 11 isDaunorubicin plus PegC and Group 13 is Daunorubicin. The approximate 10%decrease in mean relative body weight was due to daunorubicin.Example 4: The present example was conducted in a manner similar toExample 1 but mPEG-r-crisantaspase conjugates (Pegcrisantaspases) weretested in combination with other compounds. FIG. 2 shows thatPegcrisantaspase potentiates the effect of cytarbine, venetoclax, andABT-737, indicating synergy.Example 5: mPEG-r-crisantaspase conjugates (Pegcrisantaspases) wastested in combination with ABT-737 against HL-60 cell line.

Plate preparation. The stocks of the mixtures and single agents werediluted in DMSO or 0.9% sodium chloride to generate a 7-pointsdose-response series. After further 31.6 times dilution in 20 mM sterileHepes buffer pH 7.4 (reference compounds) or medium (pegcrisantaspase),5 μl of pegcrisantaspase solution, and 5 μl of reference compound wasadded to 40 μl pre-plated cells in duplicate in a 384-well assay plate.The final DMSO concentration during incubation was 0.4% in all wells.Final assay concentrations range, for the single agents, between 10 and0.01 times their IC₅₀ (10 and 0.01 IC₅₀ equivalents).

Cell proliferation assay. A cell assay stock was thawed and diluted inappropriate medium and dispensed in a 384-well plate, depending on thecell line used, at a concentration of 800-3200 cells per well in 45 μlmedium: i.e., DB: 800 cells per well; RL: 1000 cells per well; MV-4-11:1600 cells per well; KG-1, HL-60 and HT 3200 cells per well. For eachused cell line the cell density was optimized previously. The margins ofthe plate were filled with phosphate-buffered saline. Plated cells wereincubated in a humidified atmosphere of 5% CO₂ at 37° C. After 24 hours,5 μl of pegcrisantaspase solution, and 5 μl of reference compound wasadded, and plates were further incubated for another 72 hours. After 72hours, plates were cooled in 30 minutes to room temperature and 25 μl ofATPlite 1Step™ (PerkinElmer) solution was added to each well, andsubsequently shaken for 2 minutes. After 5 minutes of incubation in thedark at room temperature, the luminescence was recorded on an Envisionmultimode reader (PerkinElmer).

Controls: t=0 signal. On a parallel plate, 40 μl cells were dispensed inquadruplicate and incubated in a humidified atmosphere of 5% CO₂ at 37°C. After 24 hours, plates were cooled to room temperature in 30 minutes.5 μl DMSO-containing Hepes buffer, 5 μl 0.9% sodium chloride-containingmedium and 25 μl ATPlite 1Step™ solution were added and subsequentlymixed for 2 minutes. Luminescence was measured after 10 minuteincubation (=luminescence_(t=0)) in the dark.

Cell growth control. The cellular doubling times of all cell lines arecalculated from the t=0 hours and t=end growth signals of the untreatedcells. If the doubling time is out of specification (0.5-2.0 timesdeviating from historic average) the assay is invalidated.

Maximum signals. On each 384-well plate, the maximum luminescence wasrecorded after incubation for 72 hours without compound in the presenceof 0.4% DMSO. All equivalent wells (usually 14) were averaged. Thisaverage is defined as: luminescence_(untreated,t=72h). Dose responsecurves. Accurate single agent IC₅₀s are needed for combination analysis.For each single agent its dose-response signal was fitted by a4-parameter logistics curve using XL-fit 5 (IDBS software):

luminescence=bottom+(top-bottom)/(1+10^((logIC50−log[cpd])−hill)))

[cpd] is the compound concentration tested. hill is theHill-coefficient. Bottom and top are the asymptotic minimum and maximumof the curve.Combination Index (CI) determination. The CI is one of the most widelyused quantitative indications of synergy. The CI evaluates theconcentrations needed to achieve a fixed-effect. A CI of below 1indicates synergy. A CI of less than 0.3 indicates strong synergy. Forexample, a CI of 0.1 indicates that the combination needs a ten-foldlower concentration than expected from the single agent data, to achievethe same effect level. For instance, when a potent and less potentcompound with a CI of 0.1 are combined, the effective concentration ofthe potent compound is improved tenfold by the less potent compound.

CI is defined for a certain percentage cell viability (V), which is thesignal related to a non-exposed control:V=100%×luminescence_(treated,t=72h)/luminescence_(untreated,t=72h). Theconcentrations of the two compounds cpd1 and cpd2 needed to reach acertain percentage cell viability V in combination are then compared tothe concentrations needed as single agents:

CI_((100-V))=[cpd1]_(v)/IC_((100-V),cpd1)+[cpd2]_(v)/IC_((100-V),cpd2)

For example, [cpd1]₅₀ signifies the concentration of cpd1 in a mixturethat gives 50% viability. IC_(50,cpd1) would signify the IC₅₀ of cpd1alone. The CI is labelled by %-effect, to follow conventions, so CI₇₅signifies the CI at 25% viabilityCurve shift analysis. This analysis provides a visual confirmation ofsynergy¹. The concentrations of the mixtures of compounds 1 and 2 (cpd1and cpd2), and the single agents, were expressed in terms of IC₅₀equivalents (in ‘units’ of IC₅₀):

[mix]=[cpd1]/IC_(50,cpd1)+[cpd2]/IC_(50,cpd2)

The dose-response signal was fitted by a 4-parameter logistics curveusing XL-fit 5 (IDBS software)

luminescence=bottom+(top-bottom)/(1+10^((logX−log[mix])-hill)))

Here hill is the Hill-coefficient and X the inflection point of thecurve. Bottom and top are the asymptotic minimum and maximum of thecurve. Because [mix] is expressed in terms of IC₅₀ equivalents, thecurves of the single agents will overlap and their inflection point willlie at a value of 1. The IC₅₀ values that are used in the calculations,are those determined in parallel for the single agents.

In mixtures where synergy is absent, curves will overlap those of thesingle agents. In mixtures where there is synergy, curves will shiftleftward towards lower IC₅₀ equivalents: the mixture appears more potentthan expected on basis of the individual constituents. This is a goodindicator of synergy.

Isobolograms. An isobologram is a dose-oriented plot which revealswhether drug combinations are synergistic. It is defined at a certaineffect level, which is usually 75%. If the single agent curves do notachieve this efficacy level, the isobologram level is set at 50%, 30%,25% or 20%. If single agents do not reach the 20% effect, no isobologramis drawn. On the axis, the calculated doses of the single compounds areplotted that give the pre-set growth effect. Both points are connectedwith a straight line (additivity line). For the drug combinations, it iscalculated which dilutions give the pre-set growth effect and theconcentrations of the individual components at this point are plotted inthe isobologram. In case of an additive drug effect, the drugcombination will lie close to the additivity line. In case of synergy orantagonism, the points will lie under or above the line, respectively.

Experiments with inactive agents. In certain agreed cases, synergyexperiments are performed in the presence of ‘inactive’ agents, whichare compounds that do not give a dose-response curve as single agents,at the concentrations tested. The experiments are executed as describedabove except that the ‘inactive’ agent is added in a fixed concentrationto each well of the experiment. Because the single ‘inactive’ agentshows no effect, its contribution to CI is insignificant. CI values arethen based on the response of the active agents. Curve shift of themixture is determined compared to the other, active agent. Noisobologram is calculated. The dose-response curves with single agentsis depicted in FIG. 3 . ABT-737 has an IC50 of 835 nM and a maximaleffect at 67% while pegcrisantaspase had an IC₅₀ of 0.15 nM and amaximal effect at 88%.

Curve shift analysis: The x-axes of the single agent curves (grey anddark grey) and the mixture curves (red, orange and pink) were translatedto an IC₅₀ equivalent, based on the IC₅₀s of the single agents, and arecompared to the dose-response curves of the mixture as shown in FIG. 4 .

For dose-response curves on the mixtures on an IC₅₀ basis, all curveswere superimposed and shifts recorded. A leftward shit of the mixturescurves compared to the single agent curves (grey and dark grey)indicates synergy, a rightward shift indicates antagonism (see FIG. 5and tables below).

IC₅₀ Shifts of Mixtures Compared To Single Agents

Results using the combination of pegcrisantaspase and ABT-737 are shownbelow. CI values calculated from the mixture data, ED₇₅ corresponds to25% viability. A representative value is the average CI value for thethree mixtures at 50% viability, which is indicated in the summary.

The combination data were used to generated isobolograms as shown inFIG. 6 . An isobologram is a dose-oriented plot that reveals whetherdrug combinations are synergistic. In case of synergy, combinationpoints lie under the straight additivity line. The concentration ofpegcrisantaspase is shown in IU/mL. The additivity line (dark grey)indicates concentration combinations that would give theoreticaladditivity. Drug combinations are plotted as the red, pink and orangedots. In summary, strong synergy between pegcrisantaspase and ABT-737 inHL-60 cell line was found as shown below.

IC₅₀ IC₅₀ Average CI value Average CI value Average CI value CurveCompound (IU/mL) Therapeutic (nM) Cell line 1:1, 1:2 and 2:1* 1:1, 1:5and 5:1* 1:1, 1:10 and 10:1* shift pegcrisantaspase 0.070 ABT-747 1213HL-60 0.26 0.29 0.33 yes *Average Combination Index of the mixtures atED50; CI = 1.0: no synergy; CI < 1.0: synergy; CI < 0.3: strong synergy;CI > 1.5: antagonistic ND: not determined, tested compound had anefficacy <20% NA: not applicableExample 6: The present example was conducted in a manner similar toExample 5 but the synergy with additional anti-cancer agents indifferent cell types were tested as shown below.

AML DLBCL Cell line anti-cancer agent conclusion Cell line anti-canceragent conclusion KG-1 daunorubicin synergy DB ABT-737 synergy KG-1cytarabine synergy DB venetoclax synergy KG-1 ABT-737 strong synergy DBcarfilzomib synergy KG-1 venetoclax strong synergy DB prednisolone nosynergy KG-1 dactolisib strong synergy DB vincristine strong synergyKG-1 bortezomib synergy HT carfilzomib no synergy/antagonism KG-1carfilzomib synergy HT vincristine synergy HL-60 daunorubicin antagonismHT ABT-737 synergy HL-60 cytarabine synergy HT venetoclax no synergyHL-60 ABT-737 strong synergy RL ABT-737 synergy HL-60 venetoclax strongsynergy RL venetoclax synergy HL-60 dactolisib synergy RL carfilzomibsynergy HL-60 bortezomib antagonism RL prednisolone synergy HL-60carfilzomib antagonism RL vincristine synergy MV-4-11 daunorubicinMV-4-11 cytarabine MV-4-11 ABT-737 MV-4-11 venetoclax MV-4-11 dactolisibMV-4-11 bortezomib MV-4-11 carfilzomibExample 7: The present example was conducted in a manner similar toExample 1 but mPEG-r-crisantaspase conjugates were tested for activityagainst CNS cell lines, including for example, glioblastoma,medulloblastoma, glioblastoma multiforma and glioblastoma astrocytoma.Results are displayed in FIG. 7 . Additional experiments using differentcell lines were performed, and the results are displayed in FIG. 10 .Example 8: mPEG-r-crisantaspase conjugates (Pegcrisantaspases) incombination of additional compounds were tested against AML (acutemyeloid leukemia) and DLBCL (diffuse large B-cell lymphoma) cell linesin accordance with the methods described in Example 1. Results are shownin FIG. 14A-FIG. 14B. KG-1, HL-60 and MV4-11 are AML cell lines, and DB,HT and RL are DLBCL cell lines. The combination data withpegcrisantaspase and venetoclax showed strong synergy in the AML celllines.

Example 9

Pasylated conjugates of crisantaspases were tested against pegylated(PEG-crisantaspase) and non-pegylated (Erwinase) versions ofcrisantaspases along with E. coli L-asparaginase (Oncaspar) in multiplecell lines in accordance with the methods described in Example 1. PA-20and PA-40 are pasylated crisantaspase conjugates produced inCorynebacterium or Pseudomonas expression systems and PA-200 is apasylated fusion protein produced in Pseudomonas expression system. ThePA-20, PA-40, PA-200 and PA-400 constructs are SEQ ID NO: 2, 3, 6 and 7.Results are shown in FIG. 15 -FIG. 19 . CCRF-CEM, MOLT-4 and RS4:11 areall AML cell lines, Jurkat E6-1 is an acute T-cell leukemia cell line,HL-60 is an acute promyelocytic leukemia cell line, MV4-11 is abiphenotypic B-cell myelomonocytic leukemia cell line, THP-1 is an AMLcell line, RL is a non-Hodgin's lymphoma cell line, and H9 is a lymphomacell line.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating a disease in a patient comprising administeringto the patient an effective amount of a conjugate comprising: (i) aprotein with at least 95% sequence identity to SEQ ID NO:1, linked to a(ii) polyethylene glycol (PEG) molecule, with a molecular weight of 500to 9000 Da, wherein the disease is colon cancer.
 2. The method of claimwherein the protein has at least about 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO:
 1. 3. The method of claim wherein theprotein has 100% sequence identity to SEQ ID NO:
 1. 4. The method ofclaim wherein the PEG molecule has a molecular weight of about 5000 Da,4000 Da, 3000 Da, 2500 Da, or 2000 Da.
 5. The method of claim whereinthe conjugate has an in vitro activity of at least 60%, 65%, 70%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared toprotein when not conjugated to the PEG molecule.
 6. The method of claim1, wherein the conjugate has an L-asparagine depletion activity at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more potent thanthe protein when not conjugated to the PEG molecule.
 7. The method ofclaim wherein the conjugate depletes plasma L-asparagine levels to anundetectable level for at least about 12, 24, 48, 96, 108, or 120 hours.8. The method of claim wherein the conjugate has a longer in vivocirculating half-life compared to protein when not conjugated to the PEGmolecule.
 9. The method of claim 1, wherein the conjugate has a longert½ than pegaspargase administered at an equivalent protein dose.
 10. Themethod of claim wherein the conjugate has a t½ of at least about 58 to65 hours at a dose of about 50 μg/kg on a protein content basis, and at½ of at least about 34 to about 40 hours at a dose of about 10 μg/kg ona protein content basis, following iv administration in mice.
 11. Themethod of claim wherein the conjugate has a t½ of at least about 100 toabout 200 hours at a dose ranging from about 10,000 to about 15,000IU/m² (about 20-30 mg protein/m²).
 12. The method of claim 1, whereinthe conjugate has a greater area under the curve (AUC) compared to theprotein when not conjugated to the PEG molecule.
 13. The method of claim1, wherein the conjugate has a mean area-under-curve (AUC) that is atleast about 3 times greater than pegaspargase at an equivalent proteindose.
 14. The method of claim wherein the PEG molecule is covalentlylinked to one or more amino groups of the protein.
 15. The method ofclaim 14, wherein the PEG molecule is covalently linked to the one ormore amino groups by an amide bond.
 16. The method of claim wherein thePEG molecule is covalently linked to at least from about 40% to 100% ofaccessible amino groups of the protein or at least from about 40% to 90%of total amino groups of the protein.
 17. The method of claim 1, whereinthe conjugate has the formula:Asp-[NH—CO—(CH₂)_(x)—CO—NH-PEG]_(n) wherein Asp is the protein, NH isone or more amino groups of lysine residues and/or N-termini of the Asp,PEG is the PEG molecule, n is a number that represents at least about40% to about 100% of accessible amino groups in the Asp, and x is aninteger ranging from about 1 to
 8. 18. The method of claim wherein thePEG molecule is monomethoxy-polyethylene glycol (mPEG).
 19. The methodof claim wherein disease is a cancer the colon cancer is colonadenocarcinoma.
 20. (canceled)
 21. The method of claim wherein the coloncancer is colon carcinoma, colon adenocarcinoma, or colorectaladenocarcinoma.
 22. The method of claim 1, wherein the conjugate isadministered at an amount of about 5 U/kg body weight to 50 U/kg bodyweight.
 23. The method of claim 1, wherein the conjugate is administeredat a dose ranging from about 10,000 to 15,000 IU/m².
 24. The method ofclaim 1, wherein the administration is intravenous or intramuscular andis once per week, twice per week, or three times per week.
 25. Themethod of claim 1, wherein the conjugate is administered as monotherapy.26. The method of claim 23, wherein the conjugate is administered aspart of a combination therapy.
 27. The method of claim 25, wherein theconjugate is administered as part of a combination therapy withOncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, Venetoclax,dactolisib, bortezomib, carfilzomib, vincristine, prednisolone,everolimus, and/or CB-839.
 28. The method of claim 1, wherein thepatient receiving treatment has had a previous hypersensitivity to anEscherichia coli asparaginase or a PEGylated form thereof or to anErwinia asparaginase.
 29. The method of claim 1, wherein the patientreceiving treatment has had a disease relapse, in particular a relapsethat occurs after treatment with an Escherichia coli asparaginase or aPEGylated form thereof.
 30. The method of claim 1, wherein the coloncancer includes a mutation in a gene selected from NRAS, PTEN, ARHGAP35,MLH1, CHD4, CDKN2A, COND1, ASXL1, ERBB2, KEAP1, an ACVR1B, SMARCA4,FBXW7, MSH6, PCU2F2, PPMID, NSD1, TNFAIP3, ARID1A, CTCF, JAK1, MLL4,EP300, STIK11, SPEN, CTNNB1, SMC3, KRAS, LRP1B or a combination thereof.